(1) Field of the Invention
The invention relates to materials and methods for preparing vaccines and recombinant DNA expression products, and more particularly to genetically engineered attenuated pathogenic microorganisms that are useful for expressing antigens and other recombinant products encoded on plasmid-borne genes.
(2) Description of the Related Art
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Genetically engineered microorganisms have widespread utility and importance. One important use of these microorganisms is as live vaccines to produce an immune response. Live vaccines are most effective when they produce high levels of antigen. However, the synthesis of a high level expression of a recombinant antigen may be deleterious to the microorganism. Because of this, regulated (as opposed to constitutive) expression systems have been identified and utilized where the recombinant gene of interest is operably linked to control elements that allow expression of significant amounts of the recombinant gene only when it is induced, derepressed or activated. Examples include the cspA gene promoter, the phoA gene promoter, PBAD (in an araC-PBAD system), the trp promoter, the tac promoter, the trc promoter, xcexPL, P22 PR, mal promoters, and the lac promoter. These promoters mediate transcription at low temperature, at low phosphate levels, in the presence of arabinose, in the presence of at low tryptophan levels, and in the presence of lactose (or other lac inducers.
One important use of genetically engineered microorganisms is as a live vaccine for inducing immunity. See, e.g., U.S. Pat. Nos. 6,024,961; 4,888,170; 5,389,368; 5,855,879; 5,855,880; 5,294,441; 5,468,485; 5,387,744; 5,840,483, 5,672,345; 5,424,065; 5,378,744; 5,888,790; 5,424,065; 5,656,488; 5,006,335; 5,643,771; 5,980,907; 5,851,519; and 5,527,529, all of which are incorporated by reference. When the genetically engineered microorganism is to be utilized as a vertebrate live vaccine, certain considerations must be taken into account. To provide a benefit beyond that of a nonliving vaccine, the live vaccine microorganism must attach to, invade, and survive in lymphoid tissues of the vertebrate and expose these immune effector sites in the vertebrate to antigen for an extended period of time. By this continual stimulation, the vertebrate""s immune system becomes more highly reactive to the antigen than with a nonliving vaccine. Therefore, preferred live vaccines are attenuated pathogens of the vertebrate, particularly pathogens that colonize the gut-associated lymphoid tissue (GALT) or bronchial-associated lymphoid tissue (BALT). An additional advantage of these attenuated pathogens over nonliving vaccines is that these pathogens have elaborate mechanisms to gain access to lymphoid tissues, and thus efficient exposure to the vertebrate""s immune system can be expected. In contrast, nonliving vaccines will only provide an immune stimulus if the vaccine is passively exposed to the immune system, or if host mechanisms bring the vaccine to the immune system.
As described in U.S. Pat. No. 5,888,799, for example, pathogenic bacteria can be attenuated by introduction of mutations so that upon infection of an animal host disease symptomology is not elicited, yet the bacteria retain the ability to attach to, invade, and colonize lymphoid tissues within the animal host for a sufficient time to induce an immune response against the attenuated bacteria. These attenuated bacterial vaccine strains can be genetically engineered to express foreign antigens encoded by genes on plasmid vectors or inserted into the chromosome that are derived from heterologous pathogenic bacteria, viruses, fungi, or parasites. These recombinant attenuated bacterial vaccines can be delivered as live vaccines to mucosal surfaces in an immunized individual so that the recombinant bacteria serve as a factory within these lymphoid tissues of the immunized vertebrate, producing the foreign antigen and eliciting a primary and/or protective immune response enabling the immunized animal host to survive infection by the pathogen whose antigen is expressed by the recombinant attenuated bacterial vaccine.
Bacteria can be attenuated by introducing mutations that permit environmental regulation of surface molecule synthesis such as lipopolysaccharides in gram-negative microorganisms as affected by a gale mutation (U.S. Pat. No. 5,006,335). Bacteria can also be attenuated by introduction of mutations that impose specific nutritional requirements, such as for constituents of nucleic acids such as purines, constituents of the cell wall such as diaminopimelic acid (LAP) (U.S. Pat. No. 4,888,170), or that impose requirements for aromatic amino acids and vitamins derived therefrom, such as caused by aro mutations (U.S. Pat. No. 5,643,771). Still other means of attenuation are achieved by mutating genes affecting global regulation of other genes. Thus mutants with mutations in the genes for adenylate cyclase, cya, and the cAMP receptor protein, crp, are attenuated and immunogenic (U.S. Pat. Nos. 5,294,441; 5,389,368; 5,468,485; 5,855,879 and 5,855,880). Similarly, mutations in the phoPQ two-component regulatory system (U.S. Pat. No. 5,424,065) and mutations such as ompR U.S. Pat. No. 5,527,529), hemA Benjamin et al., 1991, Microb. Pathog. 11:289-295), and htrA (U.S. Pat. No. 5,980,907) have also been used to render bacteria attenuated yet immunogenic. All mutants of pathogenic bacteria that are attenuated are not necessarily immunogenic to the same degree. It is therefore possible to introduce mutations such as rpoS which render bacteria attenuated, but impair the ability of the attenuated bacteria to colonize lymphoid tissues, thus reducing the immunogenicity of the bacteria. See U.S. Pat. No. 6,024,961. Thus, some attenuation mechanisms hyperattenuate the vaccine, precluding the candidate vaccine from either reaching or persisting in lymphoid tissues to a sufficient extent or duration to permit induction of a protective immune response to the wild-type pathogen whose antigen is expressed by the recombinant attenuated bacterial vaccine.
Since immune responses induced to expressed foreign antigens are proportional to the levels of antigen expressed by the recombinant attenuated bacterial vaccine (Doggett et al., 1993, Infect. Immun. 61:1859-1866; Schodel et al., 1994, Infect. Immun. 62:1669-1676; Srinivasan et al, 1995, Biol. Reprod. 53:462-471), the placement of the gene for the foreign antigen on a multi-copy plasmid vector is much preferable to the insertion of the gene for the foreign antigen into the chromosome of the attenuated bacterial vaccine vector. This is because the level of foreign antigen expression is generally proportional to the number of copies of the gene for the foreign antigen expressed within the attenuated bacterial host.
Since plasmid-containing recombinant attenuated bacterial vaccines produce large amounts of antigen that provides no advantage to the vaccine, the plasmid vectors are often lost over time after immunization (Curtiss et al, 1988, Vaccine 6:155-160). In many cases, ten percent or less of the recombinant attenuated bacterial vaccine isolated from the immunized vertebrate retains the plasmid after three or four days. When this plasmid loss occurs, the immune response is directed more against the attenuated bacterial host vaccine itself rather than against the expressed foreign antigen. This problem was solved by the establishment of balanced-lethal host-vector systems as described in U.S. Pat. Nos. 5,672,345 and 5,840,483. In that system, a mutation is introduced into the chromosome of the attenuated bacterial vaccine to preclude synthesis of an essential cell wall constituent, diaminopimelic acid or DAP, which is not prevalent in the environment and is totally absent in animal tissues. In the absence of DAP, the DAP-requiring bacteria undergoes DAP-less death and lysis. The bacterium also contains a plasmid vector comprising a gene complementing the mutation in the chromosome. The plasmid-containing strain is thus able to synthesize DAP and survive in the absence of an exogenous DAP supply, as occurs in an immunized vertebrate. Colonization of internal lymphoid organs in the immunized vertebrate can then occur. One such system employs deletion mutations for the gene for xcex2-aspartate semialdehyde dehydrogenase, the asd gene, and plasmid vectors that would contain the wild-type asd gene in addition to the elements causing expression of a foreign antigen (Nakayama et al., 1988, Bio/Tech. 6:693-697; Galan et al., 1990, Gene 94:29-35). Aside from the Asd+ plasmid vector encoding the foreign antigen, these xcex94asd bacterial strains also contain attenuating mutations as described above. When orally administered, these balanced-lethal host-vector vaccines effectively attach to, invade, and colonize lymphoid tissues similar to a bacterium attenuated in the same manner but not expressing the foreign antigen. An important additional benefit of this balanced-lethal host-vector system is the absence of antibiotic resistance gene on the plasmid vector, since live vaccines are not permitted to contain such genes.
As stated above, the level of immune response to a foreign antigen is generally proportional to its level of expression by the recombinant attenuated bacterial vaccine. Unfortunately, overexpression of a foreign antigen is often toxic such that it reduces the rate of growth and therefore the ability of the attenuated bacterial vaccine to colonize lymphoid tissues. As a consequence, the immunogenicity is much diminished. For this reason, it has therefore been necessary to have a balance between the ability of the vaccine to colonize and grow in lymphoid tissues with its ability to produce the foreign antigen.
Another issue of importance in the use of recombinant attenuated bacterial vaccines is their potential, after administration to an animal or human, to be shed in feces and survive in the environment so as to potentially lead to immunization of individuals in which immunization is not desired. This issue is particularly important with agricultural vaccines that might be administered in the feed and/or drinking water or by spray such that the vaccine could persist in the environment and expose other animals to that vaccine other than the animal species desired to be immunized. We therefore designed an environmentally limited viability system (ELVS) for recombinant attenuated bacterial vaccine constructions as described in WO96/40947, incorporated herein by reference. In some embodiments of that invention, vaccine strains were constructed that, in a permissive environment, express essential genes and not express lethal genes, but upon entering a non-permissive environment, such as the ambient temperature following shedding of fecal matter, would cease expressing essential genes and commence expressing lethal genes, leading to the death of the vaccine construct. In other embodiments, the containment features of the vaccine relating to the expression of essential genes and the non-expression of lethal genes in a permissive environment, such as during growth of the vaccine strain in a fermenter, were extended for a period of time after the vaccine strain entered a non-permissive environment, such as the immunized animal host. Such delayed onset environmentally limited viability systems enable the vaccine to attach to, invade, and colonize lymphoid tissues prior to the onset of death brought about by non-expression of essential genes and expression of lethal genes. Although many examples of essential genes and lethal genes are described in WO96/40947, a preferred regulated essential gene therein is the asd gene encoding xcex2-aspartate semialdehyde dehydrogenase, an enzyme necessary for the biosynthesis of DAP, which is an essential constituent of the rigid layer of the bacterial cell wall and is not available in the environment, and especially in animal hosts. The preferred lethal genes therein are those derived from a bacterial virus that lead to lysis of the bacterium when expressed from within the cytoplasm of the microorganism. One way that biological containment is achieved in those inventions is through the employment of a runaway plasmid vector that serves as both a balanced-lethal host-vector system (to maintain the plasmid) and as an ELVS to provide biological containment. As disclosed therein, in the permissive environment (e.g., a fermenter) the bacteria would maintain a very low plasmid copy number and even turn off the expression of the plasmid-encoded foreign antigen. However, at some time after entering a non-permissive environment (e.g., the immunized animal host), the system causes plasmid copy number to increase very significantly, increasing the number of copies of the lethal genes for phage induced lysis. Since the copy number of the gene specifying the foreign antigen is increased, overproduction of the foreign antigen occurs at a time near the time when the bacterium might die by lysis to liberate the foreign antigen and thus augment the induction of an immune response.
Based on the above discussion, there is a need for a live vaccine that is able to effectively colonize the inoculated animal and grow in the lymphoid tissues without causing disease, yet still have the capacity to produce large amounts of antigen in vivo to induce an effective immune response. The present invention addresses that need through the utilization of regulated antigen delivery systems (RADS) based on the use and function of runaway vectors (RAVs).
Briefly, therefore, the inventors have succeeded in discovering that a novel Regulated Antigen Delivery System (RADS), comprising a novel runaway vector (RAV) and at least one activatible chromosome-derived repressor, in which the copy number of an extrachromosomal vector increases greatly in response to the derepression of the vector caused by the withdrawal of the activating stimulus, can be advantageously utilized in bacterial expression systems, preferably live bacterial vaccines that are attenuated derivatives of pathogenic microorganisms. The derepressible runaway characteristic of the RADS is derived from the chromosomal activatible repressors in combination with elements of the RAV. The essential elements of the RAV are (a) a first origin of replication (ori) conferring a low copy number, where the first ori preferably confers vector replication using DNA polymerase III; (b) a second ori, operably linked to a first promoter that is repressed by a chromosome-encoded repressor, wherein the second ori preferably confers vector replication using DNA polymerase I; and (c) a foreign gene, operably linked to a second promoter that is preferably also repressed by a chromosome-encoded repressor. As a vaccine, the RADS is capable of causing an effective exposure of the immunized vertebrate""s lymphoid tissues to a large dose of vector-encoded foreign gene product production in response to the withdrawal of the stimulus. Another advantage provided as a vaccine is the ability of the RADS microorganism to be grown in vitro under low copy number control, then switched to runaway conditions after vertebrate inoculation to cause an increase in antigen production in vivo. Under derepressed runaway conditions, the RADS microorganism is highly impaired due to extremely high plasmid replication activity coupled with extremely high foreign gene product production. Because of its impaired state, the derepressed RADS microorganism cannot generally survive for extended periods. The RADS therefore features an inherent containment system, in which the RADS microorganism cannot survive when not exposed to the repressor gene-activating stimulus, even in the absence of derepressible plasmid-derived phage lysis genes in the environmentally limited viability system (ELVS) as disclosed in WO96/40917.
The switch to the derepressed runaway state can be delayed after exposure of the microorganism to the derepressing environmental stimulus. In this xe2x80x9cdelayed RADS,xe2x80x9d the repressible promoters on the RAV continue their repression of the runaway condition and antigen production for a time even when the repressing stimulus is discontinued. An example of an activatible promoter that can be operably linked to a repressor on the RADS chromosome, and that is useful in delayed RADS, is the araC-PBAD promoter, which responds to arabinose. When linked in a RADS to a repressor such that the presence of arabinose represses the runaway condition, the transfer of the RADS bacteria to an environment without arabinose (such as when inoculated in a vertebrate) does not derepress the high copy number ori until arabinose that is still present inside the bacteria diffuses out or becomes metabolized by the microorganism. The delay can be advantageously increased by conferring mutations in the microorganism that eliminate its ability to metabolize the activating stimulus. This can be accomplished in the exemplified case with a mutation in the araCBAD operon to eliminate the ability of the microorganism to metabolize arabinose. The increased delay in this enhanced delay system is because the derepression to the runaway state is no longer influenced by the metabolism of the activator since the ability to metabolize the activator is eliminated. Thus, derepression is dependent only on diffusion of the activator (arabinose) out of the microorganism. Other means to alter and/or delay runaway replication and/or foreign gene expression are also disclosed.
The delay RADS is particularly useful for live bacterial vaccines because it allows time for the bacteria to colonize the vertebrate""s lymphoid tissues before switching to high copy number and producing high levels of antigen. As such, the delayed RADS is very effective in vaccines administered intranasally. When the delay is enhanced by mutations preventing metabolism of the repressor as described above, the delay is sufficient for an oral vaccine to be ingested and colonize the gut-associated lymphoid tissue (GALT) before the derepressed runaway state allows production of high amounts of antigen. Thus, high antigen levels are delivered directly to the GALT, causing a highly effective immune response.
The RADS of the present invention can be utilized in conjunction with known mutations used to attenuate the virulence of the preferred pathogenic live vaccines. The RADS is also fully compatible with plasmid maintenance systems such as the balanced lethal systems as disclosed in U.S. Pat. No. 5,672,345.
Thus, in one embodiment, the present invention is directed to a microorganism comprising a regulated antigen delivery system (RADS). The RADS comprises (a) a vector comprising (1) a site for insertion of a gene encoding a desired gene product; (2) a first origin of replication (ori) conferring vector replication using DNA polymerase III; and (3) a second ori conferring vector replication using DNA polymerase I. Further, the second ori is operably linked to a first control sequence repressible by a first repressor, and the runaway vector does not comprise a phage lysis gene. The RADS also comprises a gene encoding a first repressor operably linked to a first activatible control sequence. Preferably, the vector also comprises a gene encoding a desired gene product inserted into the site of step (a), wherein the gene encoding the desired gene product is operably linked to a second control sequence. The first control sequence and the second control sequence can be the same sequence or different sequences. Preferred repressors are LacI repressor and C2 repressor; the second control sequence can be repressible by a second repressor.
Preferably, the microorganisms described above is an attenuated derivative of a pathogenic bacterium. Also, the vector is preferably a plasmid and the desired gene product is an antigen. Most preferably, the microorganism is a Salmonella sp. A preferred activatible control sequence is araCPBAD.
The above-described microorganisms can include a balanced-lethal host-vector system consisting of a lack of a functioning essential gene on the chromosome and a recombinant functioning copy of the essential gene on the vector. The essential gene is preferably an asd gene. In one embodiment, the asd gene is inactivated by the insertion of a repressor gene operably linked to araCPBAD. The microorganisms can also comprise an inactivating mutation in a native gene that is selected from the group consisting of cya, crp, phoPQ, ompR, galE, cdt, hemA, aroA, aroC, aroD and htrA.
In the above described microorganisms, the first ori is preferably a pSC ori, and the second ori is preferably a pUC ori; the first control sequence is preferably P22 PR and the first repressor is preferably C2 repressor. Additionally, the second control sequence is preferably Ptrc and the second control sequence preferably repressible by a second repressor, which is preferably a LacI repressor. An example of a preferred runaway vector of the present invention is pMEG-771 with a gene encoding an antigen. Modifications of that runaway vector, and other exemplified vectors, is also within the scope of the invention. Examples of antigens for use in the present invention are Ery65 and SeM.
In an additional embodiment, the desired gene in the microorganism is operably linked to a eukaryotic control sequence. In these embodiments, the microorganism also preferably comprises a xcex94endA mutation.
The microorganism of the present invention can also exhibit delayed RADS characteristics. The delayed RADS characteristics are preferably conferred by an alteration selected from the group consisting of (a) a mutation that delays the loss of activator molecules by metabolism or leakage, (b) a mutation or insertion to increase repressor concentration, and (c) inclusion of a vector control sequence with binding sites for more than one repressor and/or vector sequences encoding repressor molecules that act on a vector control sequence.
The present invention is also directed to a method of producing a desired gene product. The method comprises, in order, (a) engineering a gene encoding the desired gene product into the vector of any of the above described microorganisms, wherein the microorganism comprises control sequences that represses expression of the second ori under a first environmental condition, but in which the expression of the second ori is derepressed under a second environmental condition; (b) culturing the above described microorganism under the first environmental condition; and (c) culturing the microorganism under the second environmental condition for a time sufficient to produce the desired gene product. A preferred first environmental condition comprises the presence of arabinose and a preferred second environmental condition comprises the absence of arabinose. The first environmental condition can be achieved under in vitro culture conditions and the second environmental condition can be achieved in a vertebrate. The microorganism used in this method can also comprise an inactivating deletion in the araCBAD operon and/or in the araE gene.
The present invention is also directed to a vaccine for immunization of a vertebrate, wherein the vaccine comprises any of the microorganisms described above, in a pharmaceutically acceptable carrier.
In an additional embodiment, the present invention is also directed to a method of inducing immunoprotection in a vertebrate. The method comprises administering the above vaccine to the vertebrate.
The present invention is also directed to a method of delivering a desired gene product to a vertebrate. The method comprises administering any of the above microorganisms to the vertebrate.
Among the several advantages achieved by the present invention, therefore, may be noted the provision of vectors and microorganisms for production of a desired gene product, as in a live bacterial vaccine, in which a runaway condition is effected by environmental conditions that derepress constitutive vector replication and gene product production; the provision of vaccines comprising the above microorganisms for superior stimulation of immunoprotection to the antigen gene product; the provision of methods for inducing immunoprotection to a antigen gene product by using the above vaccines; and the provision of methods for delivering a desired gene product to a vertebrate.